D.F. Visser et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 279–285
285
100
80
60
40
20
0
Immobilization of the enzyme led to an increase in stability
for EcUP and a further increase in stability for UPL8. The yields
obtained with immobilized enzymes were similar to the free
enzyme preparations at 60 ◦C and higher than the free enzymes
at 70 ◦C. Co-immobilized enzymes (PNP and UP), provided higher
yields at 70 ◦C. Reactor productivity was not equivalent to the free
enzyme systems at equal enzyme loading, indicating a potential
mass transfer limitation. Increasing the immobilized enzyme load-
ing however resulted in the high productivity observed in the free
enzyme reaction. Considering the possibility of recycling the immo-
bilized catalysts, such a system would then be more cost-effective
than the use of free enzymes. Optimization of the immobilization
method with the aim of improving activity retention will be per-
formed in future work.
0
1
2
3
4
5
6
7
8
Time (h)
Fig. 6. Yield of 5-MU (ꢀ) and guanosine conversion (ꢀ) over time by transglycosyla-
tion using either 2000 U l−1 EcUP (broken lines) or 1000 U l−1 UPL8 with equivalent
amounts of BHPNP1. Reactions were performed at 60 ◦C (EcUP) or 65 ◦C (UPL8) in
50 mM sodium phosphate buffer, pH 7.5, with 9% m m−1 guanosine and 4.6% m m−1
thymine as the starting substrate concentrations. Data averaged from triplicate
samples.
Acknowledgements
We would like to thank Dr. Moira Bode, Dr. Greg Gordon, Dr.
Petrus van Zyl, Mr. Kgama Mathiba and Dr. Dave Walwyn (ARVIR)
for their inputs. The research was financially supported by the CSIR
Young Researchers Establishment Fund.
70 ◦C compared to free enzyme systems indicating that immobi-
lization improved the thermal stability of the enzymes. The lower
productivity observed is likely due to mass transfer limitations.
An experiment was therefore performed at 1.5% m m−1 substrate
loading using 5 fold higher loading of UPL8-SZ and BHPNP1-SZ
(1000 U l−1 compared to 200 U l−1) to prove that the low produc-
tivities could be improved by higher enzyme loading. This resulted
in an increase in productivity to 4.16 g l−1 h−1 compared to 1.19 for
the same reaction using 200 U l−1 (Reactions 7 and 9 in Table 5,
respectively).
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Increasing the temperature of the reaction could increase pro-
ductivity of 5-MU production. This required a catalyst that was
more thermostable. This stability enhancement was attempted
through mutagenesis and immobilization. We have shown here
that it is possible to increase the thermal stability of E. coli UP by
directed evolution, without the need for extensive screening. The
mutation shown here increased the thermostability of the enzyme
two-fold at 60 ◦C and gave a ten-fold improvement at 70 ◦C. This
was achieved after screening fewer than 20000 clones. Small scale
experiments showed that the mutant enzyme UPL8 is a superior
catalyst for the production of 5-MU. The increase in stability of the
mutant enzyme lead to a significant (three-fold) increase in reac-
tor productivities while maintaining the high yields (75–80%) in
the free enzyme system.